PROJECT SUMMARY/ABSTRACT The ability to track electrical activity in genetically defined neurons deep in the brain has long been sought by neuroscientists to unravel the functions of neuronal circuits in health and disease. We seek to address this technical gap by developing genetically encoded voltage indicators (GEVIs), which are fluorescent proteins that report voltage dynamics as changes in brightness. GEVIs would be excellent completements to broadly-used indicators of calcium, another important information carrier in the brain. For example, GEVIs promise to report forms of neural activity that are not generally reported by calcium indicators in typical mammalian cortical neurons: hyperpolarizations, subthreshold depolarizations, and fast trains of action potentials. While these genetically encoded voltage indicators have shown early promise for detecting voltage dynamics, improvements in their brightness, photostability, and responsivity to fast voltage changes are needed for robust imaging in mammalian systems. Moreover, current GEVIs have particularly low performance under two-photon microscopy, the method of choice for deep-tissue imaging. The overall goal of this proposal is to develop a color palette of voltage indicators optimized for two-photon microscopy. We first propose to develop a new microscopy platform that can rapidly screen indicator variants by automatically monitoring and analyzing their fluorescence responses to fast voltage transients. Second, we propose to develop activity-specific GEVIs, that is, indicators that are optimized for specific forms of neural activity. Specifically, we will focus on optimizing a GEVI for detecting spikes and a GEVI for monitoring synaptic activity, that is, graded potentials about the resting membrane potential. Finally, we propose to expand the color palette of voltage indicators to enable two- photon multi-color imaging and all-optical electrophysiology. Voltage indicators with improved performance will be comprehensively characterized across all key metrics in acute brain slices and in vivo to facilitate deployment in downstream applications. We anticipate that this research project will produce voltage indicators with robust performance and with broad applicability in neuroscience, enabling voltage imaging of neuronal ensembles with cell type specificity and high spatiotemporal resolution.